An Intrinsic Alkalization Circuit Turns on Riboswitch under Manganese Stress in Escherichia coli.

The trace metal manganese in excess affects iron-sulfur cluster and heme-protein biogenesis, eliciting cellular toxicity. The manganese efflux protein MntP is crucial to evading manganese toxicity in bacteria. Recently, two Mn-sensing riboswitches upstream of and in Escherichia coli have been reported to mediate the upregulation of their expression under manganese shock. As the riboswitch is also responsive to alkaline shock administered externally, it is intriguing whether the riboswitch is also responsive to alkaline stress. Furthermore, how both manganese and alkaline pH simultaneously regulate these two riboswitches under physiological conditions is a puzzle. Using multiple approaches, we show that manganese shock activated glutamine synthetase (GlnA) and glutaminases (GlsA and GlsB) to spike ammonia production in E. coli. The elevated ammonia intrinsically alkalizes the cytoplasm. We establish that this alkalization under manganese stress is crucial for attaining the highest degree of riboswitch activation. Additional studies showed that alkaline pH promotes a 17- to 22-fold tighter interaction between manganese and the riboswitch element. Our study uncovers a physiological linkage between manganese efflux and pH homeostasis that mediates enhanced manganese tolerance. Riboswitch RNAs are -acting elements that can adopt alternative conformations in the presence or absence of a specific ligand(s) to modulate transcription termination or translation initiation processes. In the present work, we show that manganese and alkaline pH are both necessary for maximal riboswitch activation to mitigate the manganese toxicity. This study bridges the gap between earlier studies that separately emphasize the importance of alkaline pH and manganese in activating the riboswitches belonging to the - family. This study also ascribes a physiological relevance as to how manganese can rewire cellular physiology to render cytoplasmic pH alkaline for its homeostasis.